Diagnostic assay for latent matrix metallo-proteinase No. 9 and use thereof in the diagnosis of rheumatoid and inflammatory arthritis and inflammatory bowel disease

ABSTRACT

Elevated plasma levels of proMMP-9 and proMMP-9/TIMP-1 complex have been shown to correlate with and are useful in aiding the diagnosis of rheumatoid arthritis and inflammatory bowel disease; a hybridoma which produces a monoclonal antibody which specifically binds to and recognizes proMMP-9 and proMMP-9/TIMP-1 complex is disclosed and is designated mAb 277.13.

This application is a continuation-in-part of U.S. patent applicationSer. No. 07/968,554, which was filed on Oct. 29, 1992 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to assay methods for detecting levels ofspecific enzymes in biological samples. More particularly, the presentinvention relates to methods for the detection of MatrixMetallo-Proteinase No. 9 (hereinafter "MMP" when referring to the classof matrix metallo-proteinases and "MMP-9" when referring to matrixmetallo-proteinase No. 9 in particular). The inventive assay method isuseful to diagnose rheumatoid arthritis, inflammatory arthritis(including psoriatic, gout, systemic lupus erythematous, and spondylarthritis) and inflammatory bowel disease (hereinafter "IBD").

2. Description of the Related Art

MMPs are zinc-dependent endopeptidases that function in thephysiological degradation of matrix connective tissue such as collagens,gelatin, fibronectin, elastin, laminin and proteoglycan (Woessner, 1991,FASEB J., 5: 2145). MMP-9 is a 92 kDa proteinase which specificallydegrades type IV, V, X, and XI collagen, and is sometimes referred to inthe art as "96 kDa gelatinase" (Overall et al., 1991, Infect. Immun.,59: 4687).

MMP-9 plays a role in leukocyte extravasation, a process involving amultiplicity of cell-to-cell and cell-to-matrix interactions duringacute and chronic inflammatory reactions. An early response thattriggers the inflammatory cascade is the recruitment and subsequentactivation of polymorphonuclear leukocytes (hereinafter "PMN"). For PMNsto reach their targets, the basement membrane and connective tissuecollagen must be hydrolyzed. This hydrolysis is carried out in part byMMP-9.

Physiological variances in MMP levels are known. For instance,significant increases in plasma 72 kDa gelatinase (MMP-2 levels havebeen observed in women during the second half of pregnancy as comparedto early pregnancy and nonpregnant women (Zucker et al., 1992, J.Immunol. Methods, 148: 189).

Pathologically, MMPs have been identified as associated with severaldisease states. For example, anomalous MMP-2 levels have been detectedin lung cancer patients, where it was observed that serum MMP-2 levelswere significantly elevated in stage IV disease and in those patientswith distant metastases as compared to normal sera values (Garbisa etal., 1992, Cancer Res., 53: 4548). Also, using an ELISA methodology, itwas observed that plasma levels of MMP-9 were elevated in patients withcolon and breast cancer (Zucker et al., 1993, Cancer Res. 53: 140).However, these researchers did not investigate potential relationshipsamong MMP-9 plasma levels in arthritis and IBD.

Elevated levels of stromelysin (MMP-3) and interstitial collagenase(MMP-1) have been noted in synovial fluid derived from rheumatoidarthritis (hereinafter "RA") patients as compared to post-traumatic kneeinjury (Walakovits et al., 1992, Arth. Rheum., 35: 35). Hirose et al.,1992, J. Rheumat., 19: 593, demonstrated the presence of 92-96 kDa MMP-9activity and antigen in the synovial fluid of patients with inflammatoryarthritis, including RA. However, these researchers did not investigatepotential relationships among MMP-9 plasma levels in arthritis and IBD.

Increased levels of mRNA expression for collagenase type I (MMP-1) andcollagenase type IV (MMP-2) have been shown to be increased inulcerative colitis as compared to Crohn's disease and controls (Mattheset al., 1992, Gastroenterology, Abstract 661). However, plasma levels ofthese enzymes were not determined. Anthony et al., 1992,Gastroenterology, Abstract 591, demonstrated increasedimmuno-histochemical expression of the gelatinase antigen in a rabbitmodel of chronic inflammatory colitis. However, human material was notanalyzed in this study. Bailey et al., 1990, Biochem. Soc. Trans., 18:896, demonstrated increased immuno-histochemical expression of thegelatinase antigen in the intestine of patients suffering from Crohn'sdisease as compared to normal intestines. However, plasma levels of thisenzyme were not determined. Horowitz et al., 1987, Clin. Biochem., 20:79, demonstrated increased interstitial collagenase activity (MMP-1) incolonic mucosa of patients suffering IBD as compared with normal colonicmucosa. Plasma levels of MMP-9 were not determined.

None of these investigators report any relationships between MMP-9levels in plasma samples and either arthritis IA or IBD.

Bergmann et al., 1989, J. Clin. Chem. Clin. Biochem., vol. 27, pp.351-359, report on the detection of leucocyte gelatinase MMP-9 in thesynovial fluid of rheumatoid arthritis patients. However, plasma MMP-9levels in arthritis or IBD patients were not measured.

Published Japanese Patent Document 5034353 purports to relate tomonoclonal antibodies to human 92 kDa gelatinase.

SUMMARY OF THE INVENTION

We have identified antibodies which selectively recognize pro-MMP-9 andcomplexes of pro-MMP-9 with tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), with no substantial binding to active MMP-9.Herein "pro-MMP-9", when referring to the present invention, unlessotherwise indicated, shall be understood to refer to pro-MMP-9 andpro-MMP-9/TIMP-1 complexes, collectively. One of these antibodies, whichhas been designated mAb 277.13 (produced by a hybridoma cell line thathas been deposited with the American Type Culture Collection (ATCC),Rockville, Md., USA) and is described in more detail below, reacts withhigh affinity to soluble latent forms of human MMP-9. This antibody hasbeen used as a part of an immunologic detection assay which determinespro-MMP-9 levels in human biological fluids.

Our data indicate that the inventive antibodies are unique, as is theassay, which is capable of giving a quantitative measure of pro-MMP-9levels in human biological fluids. Our data further indicate thatpro-MMP-9 levels are a useful marker for providing information relevantto: (1) prognosis, (2) treatment decisions, and (3) as an indicator oftreatment efficiency in rheumatoid arthritis and other chronicinflammatory diseases and IBD.

Thus, one embodiment of the present invention relates to a method foraiding in the diagnosis of rheumatoid and inflammatory arthritis in apatient, comprising the steps of determining the amount of pro-MMP-9 ina plasma sample obtained from said patient and comparing said measuredamount of pro-MMP-9 to the mean amount of pro-MMP-9 in the normalpopulation, whereby the presence of a significantly elevated amount ofpro-MMP-9 in the patient's plasma is an indication of a rheumatoid orinflammatory arthritis condition.

A second embodiment of the present invention relates to a method formonitoring the progression of rheumatoid or inflammatory arthritis in apatient, comprising the steps of establishing a baseline value forplasma pro-MMP-9 in said patient, thereafter measuring the amount ofpro-MMP-9 in a plasma sample obtained from said patient and comparingsaid measured amount of pro-MMP-9 to said baseline value, whereby asignificantly elevated level of pro-MMP-9 indicates a deterioratingcondition while a significantly reduced level indicates an improvingcondition.

A third embodiment of the present invention relates to a method foraiding in the diagnosis of inflammatory bowel disease in a patient,comprising the steps of determining the amount of pro-MMP-9 in a plasmasample obtained from said patient and comparing said measured amount ofpro-MMP-9 to the mean amount of pro-MMP-9 in the normal population,whereby the presence of a significantly elevated amount of pro-MMPT-9 inthe patient's plasma is an indication of an inflammatory bowel diseasecondition.

A fourth embodiment of the present invention relates to a method formonitoring the progression of inflammatory bowel disease in a patient,comprising the steps of establishing a baseline value for plasmapro-MMP-9 in said patient, thereafter measuring the amount of pro-MMP-9in a plasma sample obtained from said patient and comparing saidmeasured amount of pro-MMP-9 to said baseline value, whereby asignificantly elevated level of pro-MMP-9 indicates a deterioratingcondition while a significantly reduced level indicates an improvingcondition.

Typically, in respect to all of the above methods, the amount ofpro-MMP-9 in the patient's plasma is measured by immunoassay, with animmunoassay that employs a monoclonal antibody being particularlypreferred. Particularly useful as such monoclonal antibody are thosewhich bind to the same epitope as mAb 277.13.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described with reference to thedrawings, wherein:

FIG. 1 is a graph depicting the light absorbance at various sampleconcentrations of pro-MMP-9 versus APMA-activated active enzyme usingthe present assay.

FIG. 2 is a graph depicting the light absorbance at various sampleconcentrations of various MMPs using the present assay.

FIG. 3 is a graph depicting the light absorbance at various sampleconcentrations of TIMP-1, pro-MMP-9, pro-MMP-2 and enzyme/inhibitorcomplexes.

FIG. 4 is a graph depicting a comparison of the pro-MMP-9 antigen levelsin synovial fluid samples from patients suffering from rheumatoidarthritis or inflammatory arthritis, and non-inflammatoryosteoarthritis. FIG. 5 is a graph depicting a comparison of thepro-MMP-9 antigen levels in plasma samples from patients suffering fromrheumatoid arthritis or inflammatory arthritis, and normal controls.

FIG. 6 is a graph depicting a comparison of the pro-MMP-9 antigen levelsin plasma samples from patients suffering from ulcerative colitis orCrohnjs Disease, and normal controls.

DETAILED DESCRIPTION OF THE INVENTION

The measurement of pro-MMP-9 (which term, as used herein and as statedhereinbefore, includes complexes of pro-MMP-9 and TIMP-1) can beaccomplished by any means appropriate. Typically, however, suchmeasurement will be accomplished by immunoassay, that is, by determiningthe binding of an anti-(pro-MMP-9) antibody to pro-MMP-9 in the plasmaunder assay. As is well-known in the art, the determination of suchantibody binding can be performed using a great variety of immunoassayformats.

The present invention is not limited to any particular immunoassayformat. Preferred, however, will be heterogeneous immunoassay formatssuch as sandwich immunoassay formats in which the antigen of interest isdetected by formation of a "sandwich" complex of a separation antibodyand a detection antibody. The separation antibody is immobilized orimmobilizable such as to a solid support, e.g., the walls of amicrotiter plate, a latex particle, a macrobead, and the like. Thedetection antibody is labeled or labelable, directly or indirectly, witha detectable label such as, without limitation, enzymes or enzymecofactors or substrates, chemiluminescent or fluorescent molecules,radioisotopes, and the like. The manner of detection can be any meansconventionally associated with the particular label employed, e.g., aspectrophotometer in the case of enzymes or enzyme cofactors orsubstrates, a luminometer or fluorometer in the case of chemiluminescentor fluorescent molecules, or beta or gamma counters in the case ofradioisotopes. Alternatively, the detection antibody can be detected bymeans of a labeled isotypic antibody.

The invention will now be further described with reference to thefollowing non-limiting examples:

EXAMPLE 1

Preparation of pro-MMP-9 Antibodies

Preparation of pro-MMP-9: Pro-MMP-9 was isolated modifying thepreviously described procedures of Hibbs et al. (J. Biol. Chem., 260:2493-2500, 1984) and Wilhelm et al. (J. Biol. Chem., 264: 17213-17221,1989). Briefly, neutrophil preparations were isolated from 3 or moreunits of freshly drawn whole blood obtained from the New York BloodCenter, Inc. (New York, N.Y.). Cells were resuspended in phosphatebuffered saline (PBS) containing 100 ng/ml phorbol myristate acetate(PMA) in the presence of 50 mM diisopropylfluorophosphate (DFP), 1 μg/mlleupeptin and aprotinin, and 1 mg/ml catalase for 1 hr at 37° C.Supernatants were collected by centrifugation (300×g) and the sampleswere frozen at -70° C. All chromatographic methods were performed at 4°C. Thawed samples were concentrated 5-fold using an Amicon chamberequipped with a YM-10 membrane. The concentrate was pressure dialyzedagainst 0.02 M Tris-HCl, 0.1 M NaCl, 1 mM CaCl₂, 1 μM ZnCl₂, 0,001%Brij-35, 0.02% sodium azide (NaN₃), pH 7.5 and applied to DEAE ionexchange chromatography resin which was previously equilibrated with thesame buffer at a flow rate of 0.4 ml/min. The column was extensivelywashed with the same buffer and gelatinase was eluted as 4 ml fractionsfrom the column with 0.02 M Tris-HCl, 0.5 M NaCl, 1 mM CaCl₂, 1 μMZnCl₂, 0.001% Brij-35, 0.02% NaN₃, pH 7.5. Gelatinase containingfractions were observed by gelatin zymography, loaded onto a gelatinagarose affinity resin and washed with the same buffer. Gelatinaseactivity was eluted at a flow rate of 1 ml/min from the column as 1 mlfractions with 0.02 M Tris-HCl, 1 M NaCl, 1 mM CaCl₂, 1 μM ZnCl₂, 0.001% Brij-35, 0.02% NaN₃, pH 7.5 containing 10% dimethyl sulfoxide (DMSO).The fractions containing gelatinase activity were pooled and dialyzedagainst 0.005 M Tris-HCl, 5 M NaCl, 0.5 mM CaCl₂, 0.1 μM ZnCl₂, 0.001%Brij-35, pH 7.4. The protein content associated with material wasdetermined with a micro-BCA assay (Pierce, Rockford, Ill.), lyophilizedand reconstituted to a desired working concentration (100 μg/ml).

The preparation of pro-MMP-9 specific polyclonal antibodies:

Purified latent form of human neutrophil MMP-9 (50 μg/animal) wasemulsified with an equal volume of complete Freund's adjuvant. Thismixture was administered to New Zealand White rabbits at multipleintradermal sites (30-50 sites) with 0.05 ml/site. Secondaryimmunizations were carried out on days 14, 21, 42 and 63 using MMP-9 (50μg/animal) emulsified with an equal volume of incomplete Freund'sadjuvant. Bleeds were taken at day 0, 28, 49 and 70 to determineantibody response.

The preparation of pro-MMP-9 specific monoclonal antibody (mAb 277.13):

Balb/c mice were immunized by intraperitoneal injection with purifiedlatent form of human neutrophil MMP-9 (15 μg/animal) emulsified with anequal volume of complete Freund's adjuvant. Secondary immunizations werecarried out on days 21 and 49 using MMP-9 (10 μg/animal) emulsified withan equal volume of incomplete Freund's adjuvant. After an additionalseven days, the mice were immunized intravenously with 5 μg of immunogenand spleens were removed for fusion three days later.

Somatic cell hybrids were prepared by the method of Herzenberg andMilstein (Handbook of Exp. Immunol., ed. Weir, D., Blackwell Scient.Public., 25.1-25.7, 1978) with some modifications (Lerner et al., 1980,J. Exp. Med., 152: 1085-1101). The non-immunoglobulin secreting mousemyeloma cell line p3X63-Ag8.653 (Af8, ATCC No. CRL 1580) was cultured inRPMI 1640 medium containing 20% fetal bovine serum, 2 mM glutamine, 50U/ml penicillin, and 50 μg/ml streptomycin. Single cell suspensions ofspleen cells were prepared from immunized mice and mixed at a 2:1 ratiowith Ag8 cells in serum-free RPMI 1640 medium. This cell mixture wasfused by the dropwise addition of prewarmed 45% (w/v) polyethyleneglycol-1450 (Eastman Kodak Co., Rochester, N.Y.) and subsequent dilutionwith 10 ml serum-free RPMI 1640 medium. After fusion, cells (1×10⁶cells/well/ml) were seeded onto 24 well plates and hybrid cells wereselected by hypoxanthine-aminopterin-thymidine supplemented culturemedium. Hybridomas were screened for the desired antibody synthesisusing enzyme linked immunosorbent assay (ELISA) and cell lines secretingantibodies of interest were cloned at least twice by limiting dilutionusing culture medium supplemented with 5 U/ml human recombinant IL-6(Genzyme, Boston, Mass.).

Culture supernatants of hybridomas were screened for the presence ofanti-pro-MMP-9 antibody using ELISA assays. Purified human neutrophilpro-MMP-9 (25 ng/well) was adsorbed to Immunolon I plates (Dynatech,Cambridge, Mass.) by overnight incubation at 4° C. in 50 μl 0.01 Msodium carbonate pH 9.5. Wells were blocked with PBS and bovine serumalbumin (BSA), sequentially incubated with 100 μl of hybridomasupernatant, washed, and incubated with 100 μl of a 1:1000 dilution ofperoxidase labeled affinity purified goat antimouse IgG (Kirkegaard andPerry Laboratories, Gaithersburg, Md.). Bound peroxidase was detectedusing tetramethylbenzidine according to the manufacturer's instruction(Kirkegaard and Perry Laboratories, Gaithersburg, Md.). Isotypes ofpositive hybrid supernatants were determined using an ELISA sandwichassay. Monoclonal antibodies were purified from ascites fluid using aprotein A Sepharose column and the MAPs buffer system (BioradLaboratories, Richmond, Calif.).

EXAMPLE 2

Pro-MMP-9 Immuno-Capture Sandwich Assay Development

Based upon ELISA and Western analysis, the antibodies- both polyclonaland monoclonal- recognized Pro-MMP-9 specifically, but not other MMPs ortissue inhibitor of matrix metalloproteinase (TIMP). By titrating humanneutrophil pro-MMP-9 using various dilutions of primary and secondaryantibody, optimal conditions for the MMP-9 immuno-capture sandwich ELISAwere established.

96-well Immunolon I microtiter plates were coated with mAb 277.13 (1μg/well) (Example 1) and incubated for 18 hrs at 4° C. in 0.01 M sodiumcarbonate pH 9.5. The plates were washed 5 times with 20 mM Tris HCl,0.15 M NaCl, 0.05% Tween-20, pH 7.4 (washing buffer) and incubated for 1hr at room temperature with 5% BSA in the same buffer to blocknonspecific protein binding to assay wells.

The BSA was removed and the plates were washed with washing buffer andthe standard human neutrophil pro-MMP-9 or patient samples were addedfor 2 hrs at room temperature. Samples were diluted in wash buffercontaining 0.05% Tween-20 along with 1% BSA. The plates were washed withwash buffer (5 times) and incubated for 1 hr at room temperature withthe rabbit polyclonal antisera (Example 1) at a 1:2000 dilution in washbuffer containing 1% BSA. This antisera was washed from the plates andthe plates were then incubated with alkaline phosphatase-labeled mouseanti-rabbit mAb (Sigma Chemical Co., St. Louis, Mo.) for 1 hr at roomtemperature in wash buffer containing 1% BSA (dilution was 1:1000,mAb:1% BSA).

The plates were again washed 5 times and incubated with the substratep-nitrophenyl phosphate (1 mg/ml) in 0.1 M glycine, 1 mM MgCl₂ and 1 μMZnCl₂, pH 10.4. The absorbance at 405 nm was measuredspectrophotometrically in an automated plate reader. The assay has ahalf maximal detection level of approximately 10 ng/ml with a log-linearrange of detection of 0.3-100 ng/ml (FIG. 1).

FIG. 1 shows that the inventive assay detects pro-MMP-9 (MW of 92-96kDa), but does not detect p-aminophenyl mercuric acetate (APMA)activated active MMP-9 (MW of 83 or 70 kDa). Data represent the meanvalues of triplicate determinations; S.D. was less than 10% of the mean.

FIG. 2 shows that the inventive antibody is selective for pro-MMP-9 andfails to recognize human PMN collagenase (MMP-8), interstitialcollagenase (MMP-1), and human stromelysin (MMP-3) at concentrations upto 50 ng/ml. The inventive assay also fails to recognize purifiedpro-MMP-2 and MMP-2/TIMP complexes. However, the inventive antibody willrecognize pro-MMP-9/TIMP-1 complexes at a similar affinity as pro-MMP-9alone (FIG. 3). Data represent the mean values of triplicatedeterminations; S.D. was less than 10% of the mean.

EXAMPLE 3

Analysis of pro-MMP-9 in Human Synovial Fluid of Arthritis Patients

Human synovial fluid derived from patients with rheumatoid arthritis(n=31), patients with osteoarthritis (n=9), inflammatory arthritispatients which included IBD (n=3), gout (n=1), psoriatic arthritis(n=4), and spondylarthritis (n=3), were analyzed for pro-MMP-9expression using the immuno-capture sandwich ELISA assay (Example 2).Detection in the linear range of the assay required a 1:50-1:1000dilution of each respective sample. FIG. 4 shows the results ofquantitation of latent MMP-9 in the synovial fluid from patients withosteoarthritis (OA), inflammatory arthritis (IA), and rheumatoidarthritis (RA). Synovial fluids from patients with RA and from patientswith OA were diluted and analyzed using the immuno-capture ELISA assay.The mean values ± standard error of 0.07±0.02 Bg/ml for the OA groupwere observed as compared to RA samples which displayed latent MMP-9levels of 6.7±2.6 Bg/ml and IA levels were 2.4±0.7 μg/ml. Thesedifferences were observed to be significant (Bonferroni modification ofa two-tailed Studentjs t-Test for unpaired values). Asterisks denotesignificance P<0.05.

These data indicate that RA synovial fluids display over a 60-foldelevation in pro-MMP-9 levels, while synovial fluids derived from IApatients displayed a 34-fold elevation in pro-MMP-9 levels above OApro-MMP-9 levels.

EXAMPLE 4

Determination of pro-MMP-9 in Human Plasma of Arthritis Patients

Human plasma derived from patients with rheumatoid arthritis (n=17),inflammatory arthritis (n=9) and from normal individuals (n=60) wereanalyzed for pro-MMP-9 expression using the immuno-capture sandwichELISA assay (Example 2). Detection in the linear range of the assayrequired a 1:100-1:1000 dilution of each respective sample. Normalplasma (n=60) demonstrated pro-MMP-9 levels of 0.56±0.1 μg/ml ascompared to RA samples (n=17) which displayed pro-MMP-9 levels of3.5±0.8 μg/ml and IA pro-MMP-9 levels of 0.25±0.1 μg/ml. Thesedifferences were noted to be significant using a Bonferroni modificationof a two-tailed student's t-test for unpaired values.

These data indicate that RA plasma displays over a 6-fold elevation inlatent MMP-9 levels as compared to normal plasma. FIG. 5 graphicallydepicts these results, showing the quantitation of latent MMP-9 in theplasma obtained from patients with RA, IA, and normal individuals. Barsdepict the mean values ± standard error, as stated above. Asterisksdenote significance P<0.05.

EXAMPLE 5

Analysis of pro-MMP-9 in Human Plasma of Ulcerative Colitis and Crohn'sDisease Patients

Human plasma derived from ulcerative colitis and Crohn's diseasepatients was analyzed for pro-MMP-9 expression using the immuno-capturesandwich ELISA assay (Example 2). Pro-MMP-9 antigen detection in thelinear range of the assay required a 1:50-1:1000 dilution of eachrespective plasma sample. Plasma derived from patients with moderate tosevere ulcerative colitis (n=9) based on physical diagnosis,demonstrated latent MMP-9 levels of 2.9±0.4 μg/ml compared to normalcontrol samples (n=60) which displayed latent MMP-9 levels of 0.56±0.1μg/ml (FIG. 6). Plasma obtained from ulcerative colitis with no or milddisease had pro-MMP-9levels of 1.4±0.3 μg/ml. In addition, plasmaobtained from patients with moderate to severe Crohn's disease (n=13)had latent MMP-9 levels of 2.5±0.5 μg/ml. Crohn's patients with no ormild disease (n=29) exhibited pro-MMP-9 levels of 1.6±0.3 μg/ml. Thesedata indicate that both ulcerative colitis and Crohn's disease plasmasamples derived from patients with moderate/severe disease activitydisplay a significant 4- to 5-fold elevation in latent MMP-9 levelscompared to control plasma samples. This increase in pro-MMP-9 wasreduced in both cases to a 2- to 3-fold elevation in the no or milddisease activity group compared to control plasma samples displaying acorrelation with clinical disease status.

In FIG. 6, the mean values ± standard error are shown for eachrespective group. Differences amongst the groups were analyzed using theBonferroni modification of a two-tailed Student's t-Test for unpairedvalues. Asterisks denote significance P<0.05: * different from control.

It will be appreciated that the instant specification and claims are setforth by way of illustration and not limitation, and that variousmodifications and changes may be made without departing from the spiritand scope of the present invention. The claims are intended to coversuch modification and changes either literally or pursuant to thedoctrine of equivalents.

We claim:
 1. A hybridoma, wherein the hybridoma produces a monoclonalantibody which specifically binds proMMP-9 and proMMP-9/TIMP-1 complexand the monoclonal antibody is designated mAB 277.13.
 2. The monoclonalantibody mAb 277.13 which specifically binds proMMP-9 andproMMP-9/TIMP-1 complex.
 3. A method for aiding in the diagnosis ofrheumatoid arthritis in a patient, comprising the steps of determiningthe amount of pro-MMP-9 in a plasma sample obtained from said patientand comparing said determined amount of pro-MMP-9 to the mean amount ofplasma pro-MMP-9 in the normal population, whereby the presence of anelevated amount of pro-MMP-9 in the patient's plasma is an indication ofincreased probability of rheumatoid arthritis.
 4. The method of claim 4wherein the amount of pro-MMP-9 in the patient's plasma is measured byimmunoassay.
 5. The method of claim 4 wherein the immunoassay employs amonoclonal antibody.
 6. The method of claim 5 wherein said monoclonalantibody binds specifically to an epitope present in pro-MMP-9 andpro-MMP-9/TIMP-1 complexes.
 7. The method of claim 6 wherein saidmonoclonal antibody mAb 277.13.
 8. A method for monitoring theprogression of rheumatoid arthritis in a patient, comprising the stepsof establishing a baseline value for plasma pro-MMP-9 in said patient,thereafter measuring the amount of pro-MMP-9 in a plasma sample obtainedfrom said patient and comparing said measured amount of plasma pro-MMP-9to said baseline value, whereby an elevated level of plasma pro-MMP-9indicates a deteriorating condition while a reduced level indicates animproving condition.
 9. The method of claim 8 wherein the amount ofpro-MMP-9 in the patient's plasma is measured by immunoassay.
 10. Themethod of claim 9 wherein the immunoassay employs a monoclonal antibody.11. The method of claim 10 wherein said monoclonal antibody bindsspecifically to an epitope present in pro-MMP-9 and pro-MMP-9/TIMP-1complexes.
 12. The method of claim 11 wherein said monoclonal antibodyis mAb 277.13.
 13. A method for aiding in the diagnosis of inflammatorybowel disease in a patient, comprising the steps of determining theamount of pro-MMP-9 in a plasma sample obtained from said patient andcomparing said determined amount of plasma pro-MMP-9 to the mean amountof plasma pro-MMP-9 in the normal population, whereby the presence of anelevated amount of pro-MMP-9 in the patient's plasma is an indication ofan inflammatory bowel disease condition.
 14. The method of claim 13wherein the amount of pro-MMP-9 in the patient's plasma is measured byimmunoassay.
 15. The method of claim 14 wherein the immunoassay employsa monoclonal antibody.
 16. The method of claim 18 wherein saidmonoclonal antibody binds specifically to an epitope present inpro-MMP-9 and pro-MMP-9/TIMP-1 complexes.
 17. The method of claim 16wherein said monoclonal antibody is mAb 277.13.
 18. A method formonitoring the progression of inflammatory bowel disease in a patient,comprising the steps of measuring a baseline value for plasma pro-MMP-9in a plasma sample obtained from said patient and comparing saidmeasured amount of pro-MMP-9 to said baseline value, whereby an elevatedlevel of pro-MMP-9 indicates a deteriorating condition while a reducedlevel indicates an improving condition.
 19. The method of claim 18wherein the amount of pro-MMP-9 in the patient's plasma is measured byimmunoassay.
 20. The method of claim 19 wherein the immunoassay employsa monoclonal antibody.
 21. The method of claim 20 wherein saidmonoclonal antibody binds specifically to an epitope present inpro-MMP-9 and pro-MMP-9/TIMP-1 complexes.
 22. The method of claim 21wherein said monoclonal antibody is mAb 277.13.